A New Species of Mayfly (Ephemeroptera: Trichorythidae) from Mindanao Island, Philippines and Association of Life Stages Using DNA Barcode*
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ARTICLE | Philippine Journal of Systematic Biology A New Species of Mayfly (Ephemeroptera: Trichorythidae) from Mindanao Island, Philippines and Association of Life Stages Using DNA Barcode* Leocris S. Batucan Jr.1,2, Olga M. Nuñeza2, Reagan Joseph T. Villanueva3 and Chung-Ping Lin1,4 ABSTRACT KEY WORDS : A new mayfly species, Sparsorythus buntawensis sp. nov. (Tricorythidae) from Mt. Malindang Layawan River of Mt. Malindang in Mindanao Island, the Philippines is described based Macroinvertebrate on nymphal and adult morphologies. Sparsorythus buntawensis sp. nov. differ from all Sparsorythus known members of the genus primarily in the deeper cleft of the hypopharynx and wider Freshwater distance between compound eyes. Conspecific specimens of various life stages and Layawan River sexes of this new mayfly were associated using DNA barcode. INTRODUCTION Mindanao Island of the Philippines, a previously unknown mayfly species have been discovered, a new Sparsorythus The species diversity of mayflies (Insecta: Ephemeroptera) in (Tricorythidae). The Tricorythidae are widespread in South- the Philippines, like in the whole Oriental region, is largely east Asia and the Indian subcontinent, and currently underestimated due to insufficient collecting effort and the encompass eight described species (Barber-James et al., region being largely “unknown lands” (Barber-James et al., 2013) within the genus Sparsorythus. Recently revised by 2008). Hubbard & Pescador (1978), cataloguing only 20 Sroka & Soldán (2008) based on Ulmer’s (1913) Philippine mayfly species, suggested that the low species Tricorythus jacobsoni from Java Island, Sparsorythus is number in the archipelago did not necessarily reflect a low considered as phylogenetically derived within the species diversity but may be caused by sporadic and Tricorythidae due to its morphological apomorphies. inadequate taxonomic works. From the first collecting record Sparsorythus differs from other tricorythids by its reduced made by Walker (1853) in Luzon to the most recent maxillary palps in nymphs, fully merged penial lobes publication of five new species by Braasch (2011) in Palawan, without auxiliary processes, and a well–developed only a total of 37 mayfly species were described from the paracercus in the imagines (Sroka & Soldán, 2008). Philippine archipelago. They are classified into eight families: Ulmer’s (1924) record of Sparsorythus jacobsoni in Luzon Baetidae, with 11 species from five genera; Caenidae, with along with Sroka & Soldán’s (2008) described yet unnamed four species from two genera; Heptageniidae, with 16 species Sparsorythus sp. 4 from Mt. Apo of Mindanao Island are from six genera; Prosopistomatidae, with two species from a the only two currently recognized tricorythids in the single genus; and one species each for Ephemeridae, Lepto- Philippines. phlebiidae, Teloganodidae, and Tricorythidae. Based on the newly collected materials from Layawan From a recent survey of aquatic insects conducted in River of Mt. Malindang on Mindanao Island, this study de- scribed one new mayfly species, Sparsorythus buntawensis 1 Department of Life Science, National Taiwan Normal University, No. 88, Sec. 4, Tingzhou Rd., Taipei 11677, Taiwan; sp. nov. (Tricorythidae). We associated sexes and life 2 Department of Biological Sciences, Mindanao State University – stages of conspecifics using DNA barcode (Gattolliat et al., Iligan Institute of Technology, 2015; Webb et al., 2012; Zhou et al., 2009; Ball et al., A. Bonifacio Ave., Tibanga, Iligan City 9200, Philippines; 2005). 3 Forestal Healing Homes and Therapeutic Milieu, Davao City 8000, Philippines. 4 Correspondence: [email protected]. MATERIALS AND METHODS * Article Details Submitted : 25 April 2016 Layawan River is characterized by a rocky riverbed with Accepted : 12 October 2016 large boulders, dense riparian vegetation upstream, and © Association of Systematic Biologists of the Philippines Vol. 10 | 6 1 Philippine Journal of Systematic Biology | Batucan et al.: A New Species of Mayfly (Ephemeroptera: Trichorythidae) Figure 1: (a) The habitat of Layawan River, and (b) a modified 3D map of Mt. Malindang (ICRAF, 2013) showing the river system and sampling sites. Inset is Mindanao Island located in southern Philippines, the region in red is the location of Mt. Malindang Range. farmlands downstream (Fig. 1a). This river drains into the minute of extension at 72 ºC ( 35 cycles), and 10 minutes Oroquieta watershed and is one of the major rivers in the Mt. of final extension at 72 ºC. PCR products were visualized Malindang Range Natural Park (Hansel et al., 2006), the only on 1.5% agarose gels and purified by shrimp alkaline representative natural forest remaining in the Zamboanga phosphatase/exonuclease I, and then subjected to DNA Peninsula biogeographic zone of Mindanao Island (Arances, sequencing using the BigDye® terminator 3.1 sequencing 2006) and newly designated as an ASEAN Heritage Park. kit on an ABI 3730XL DNA Analyzer. The region where the river lies has a Type II Philippine climate characterized by no dry season with very pronounced The forward and reverse COI sequences were assembled rainy season from December to February (PAGASA, 2015). using Seqman v7.1.0 (DNASTAR, Madison, USA). All Ten sites (Fig. 1b) ranging from 73 to 1,215 m elevation along metadata and sequences >600 bps were uploaded to the the stretch of Layawan River were sampled. Two sampling Barcode of Life Database (BOLD, www.boldsystems.org) efforts were conducted, October 1–6, 2013 and April 13– 18, and submitted to GenBank (Table 1). Sequence analyses 2014. Nymphs were collected manually while adults were were carried out using MEGA 6 (Tamura et al.., 2013). collected through LED light traps. All field–collected Pairwise distances were calculated using Kimura–2– specimens were stored in 90% alcohol, then transferred to parameter model with pairwise deletion for gap handling 95% alcohol in the laboratory, and finally stored at –20 ºC and 1000 bootstrap replications to determine the genetic freezers. Nymphal specimens were carefully dissected and distance between conspecific individuals. slide–mounted using Bioquip Euparal mounting medium. Morphological observations and imagery were made using a Nikon EOS700d digital camera mounted on an Olympus TAXONOMY SZ61 stereomicroscope with 10 to 40x magnification. From the images captured, measurements of mayfly body parts Order Ephemeroptera were made using ImageJ v1.46r software (National Institutes Family Tricorythidae Lestage, 1942 of Health, Bethesda, MD, USA). Genus Sparsorythus Sroka & Soldan, 2008 Type species Sparsorythus bifurcatus Genomic DNA was extracted from the legs of the insects Sroka & Soldan, 2008 using a standard phenol–chloroform protocol (Lin & Wood, 2002). To amplify the fragments of cytochrome c oxidase subunit I (COI) gene of animal DNA barcode, the forward Sparsorythus buntawensis Batucan, Nuñeza, & Lin sp. nov. LCO1490 and reverse HCO2198 primer (Folmer et al., 1994) (Figs. 2–17). were used for the Polymerase Chain Reaction (PCR). Each PCR reaction contained 1 µL of DNA, 1 µL of Taq Nymph Holotype: body generally light brown to pale yellow polymerase, 2 µL of forward and reverse primer, 4 µL of with black dorsal and ventral markings (Fig. 2). Margins of dNTPs, 5 µL of buffer, and 35 µL of double–distilled water. head, abdomen, and legs yellow to pale yellow. Body The PCR procedure included: one minute of denaturation at length 5–6 mm. Cerci & paracercus 1.2 and 1.3 times as 94 ºC, followed by 45 seconds of annealing at 53 ºC, one long as body. © Association of Systematic Biologists of the Philippines Vol. 10 | 7 x Philippine Journal of Systematic Biology | Batucan et al.: A New Species of Mayfly (Ephemeroptera: Trichorythidae) Figure 2: Sparsorythus buntawensis sp. nov. (a) nymph, (b) female adult, and (c) male adult. Scale ½ mm. Head: Width to length ratio 1:1.4 (Fig. 3). Male head (Fig. 3a) width is 2:1, 2.1:1, and 2.2:1 for fore, middle, and hind legs, with median emargination posteriorly, more ovate than that of respectively. Fore femora (Fig. 6a) with black pointed setae female (Fig. 3b). Eyes black, ocelli black and smooth–edged, running halfway along depression on upper surface, triangle to bean–shaped. Compound eyes of males and directed apically and interspersed with short yellow setae, females approximately of same size. Ratio of distance upper margin with setae medially subequal in length as between compound eyes and eye width 5.2:1. upper surface setae, setal length and point variable. Ventral margin of fore tibia (Fig. 6a) with fine long hairs and one Mouthparts: Labrum (Fig. 4a) twice wider than long. stout apical pointy setae, dorsal margin with shorter and Hypopharynx (Fig. 4b) 1.3 times as wide as long with a deep thinner hairs. Dorsal surface of middle and hind femora median cleavage. Right prostheca (Fig. 4c) notched, (Fig. 6c) with very small stout setae, absent in depression. triangular, with several short pointed teeth and ½ the length of Mid- and hind femora longer than tibia. Ventral margin of inner incisor. Left prostheca (Fig. 4d) rod–like, pointed and middle tibia (Fig. 6b) with small and blunt setae, apex with notched at apex, ¾ the length of outer incisor. One stout two pointed setae. Dorsal margin of middle and hind setae near base of left prostheca, as long as right prostheca. femora (Fig. 6b, 6c) with stout and long blunt setae, Maxilla (Fig. 4e) without palps. Labial plate (Fig. 4f) without irregularly alternating in length, running medial distally. invagination at mid-anterior margin. Numerous basal setae on Dorsal margin of hind tibia (Fig. 6c) have similar setae but labial plate as long as lateral setae at second segment of more pointy ends. Caudal filament segments (Fig. 7) labial palp. surrounded with short, thin setae having maximum length approximately 1/3 of segment. Thorax and Abdomen: Gills (Fig. 5) present from segments II to VI, dorsal lamellae on segment VI (Fig. 5e) with tiny, short, Male Imago Paratype: Body length 4–5 mm. General marginal bristles. Legs (Fig. 6) robust. Length ratio (femur: coloration light black to gray. Cerci twice longer than body, tibia: tarsus): foreleg (Fig.